Murine r spondin 1

Manufactured by Thermo Fisher Scientific

Sourced in United Kingdom

Murine R-spondin-1 is a recombinant protein that functions as a Wnt pathway agonist. It is a critical regulator of stem cell self-renewal and differentiation.

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Lab products found in correlation

N acetylcysteine, by Merck Group (2 mentions) Hepes, by Thermo Fisher Scientific (2 mentions) Glutamax, by Thermo Fisher Scientific (2 mentions) Doxycycline, by Merck Group (1 mentions) D8437, by Merck Group (1 mentions) Normocyn, by InvivoGen (1 mentions) 4 oht, by Merck Group (1 mentions) Chir99021, by Thermo Fisher Scientific (1 mentions) B27 supplement, by Thermo Fisher Scientific (1 mentions) Nicotinamide, by Merck Group (1 mentions) Murine noggin, by Thermo Fisher Scientific (1 mentions) Valproic acid vpa, by Bio-Techne (1 mentions) Eclipse ts100 f microscope, by Nikon (1 mentions) Epidermal growth factor (egf), by Thermo Fisher Scientific (1 mentions) Chir99021, by Merck Group (1 mentions) Ldn193189, by Merck Group (1 mentions) Y 27632, by Selleck Chemicals (1 mentions) Penicillin, by Corning (1 mentions) Streptomycin, by Corning (1 mentions)

3 protocols using murine r spondin 1

Murine Small Intestinal Organoid Culture

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Around 100 freshly isolated crypts were counted and embedded in 50 μl of undiluted Matrigel (356231; Cultek) and culture DMEM/F12 media (D8437; Sigma-Aldrich) supplemented with Pen/Strept, 1× (2 mM) Glutamax (35050038; Gibco or Life Technologies/Thermo Fisher Scientific), 10 mM Hepes (15630049; Gibco or Life Technologies/Thermo Fisher Scientific), 2 mM N-acetyl cysteine (A8199-10G; Sigma-Aldrich), 1× B27 supplement (17504-044; Life Technologies), 10 mM Nicotinamide (N0636-100G; Sigma-Aldrich), 50 ng/ml of recombinant mEGF (PMG8044; Gibco), 100 ng/ml of recombinant Nogging (250-38; PeproTech), 3.5 μM CHIR99021 (GSK3 inhibitor; 2520691; PeproTech), 1 μg/ml murine R-spondin 1 (120-38; Peprotech), 10 μM Y-27632 inhibitor (1293823; PeproTech), and 1% Normocyn (ant-nr-1; InvivoGen; Barriga et al., 2017 (link)). Notably, CHIR99021 (GSK3 inhibitor; 2520691; PeproTech) was kept in culture for 12 h to establish the culture, and the medium was replaced without this inhibitor. The media was changed every 3 d. Depending on the experiment, the crypt culture media was supplemented with 1.5 μg/ml doxycycline (D9891; Sigma-Aldrich) or 1 μM of 4-OHT (H6278; Sigma-Aldrich). Organoids were paraffin-embedded and processed for IF and IHC following the protocols described below.

Chaves-Pérez A., Santos-de-Frutos K., de la Rosa S., Herranz-Montoya I., Perna C, & Djouder N. (2022). Transit-amplifying cells control R-spondins in the mouse crypt to modulate intestinal stem cell proliferation. The Journal of Experimental Medicine, 219(11), e20212405.

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Directed Differentiation of Intestinal Organoids

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Based upon methods from previous work [20 (link), 21 (link)], we directed differentiation of crypts into organoids enriched in ISCs, ENTs, GOBs, and PANs. Briefly, small molecules (Wnt and Notch activators/inhibitors) including CHIR99021 (3 μM; Stemgent, Cambridge, MA), 2 mM valproic acid (VPA; Tocris Bioscience, United Kingdom), 2 μM C59 (Stemgent, Cambridge, MA), and 10 μM DAPT (Tocris Bioscience, United Kingdom) were added to the base crypt culture medium (CCM) containing EGF (50 ng/ml; Life Technologies), murine R-spondin-1 (500 ng/mL; PeproTech), and murine Noggin (100 ng/mL; PeproTech). Organoids in CCM with all cell types represented are referred to as typical (TYP). ENT, GOB, and PAN organoids are differentiated from ISC organoids, and characterized at ~3 days after initiation of differentiation when biomarker expression becomes stable [20 (link)]. Bright-field images were obtained using an ECLIPSE TS100-F microscope (Nikon, Japan).

Pearce S.C., Al-Jawadi A., Kishida K., Yu S., Hu M., Fritzky L.F., Edelblum K.L., Gao N, & Ferraris R.P. (2018). Marked differences in tight junction composition and macromolecular permeability among different intestinal cell types. BMC Biology, 16, 19.

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Enteroid Monolayer Differentiation Protocol

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Organoid basal media (OBM) consists of Advanced DMEM/F12 with non-essential amino acids and sodium pyruvate (Fisher Scientific #12634–028) containing 1x N-2 (Fisher Scientific #17502–048), 1x B-27 (Invitrogen #17504–044), 10 mM HEPES (Invitrogen #15630080), 1x GlutaMAX (Invitrogen #35050–061), 1 μM N-acetylcysteine (Sigma Aldrich #A9165), 100 U/mL penicillin and 100 μg/mL streptomycin (Corning #30–002).
For initial seeding, enteroid monolayers were maintained in OBM supplemented with 3 μM CHIR-99021 (Sigma Aldrich #SML1046), 50 ng/mL murine EGF (Invitrogen #PMG8043), 1 μM LDN-193189 (Sigma Aldrich #SML0559), 500 ng/mL murine R-spondin-1 (Peprotech #315–32), and 10 μM Y-27632 (Selleck Chemicals #S1049).
4 hours after initial seeding, media was changed into OBM supplemented with 50 ng/mL murine EGF, 100 ng/mL murine Noggin, and 500 ng/mL murine R-spondin-1. Perturbations applied in the studies described here were all applied in the background of this medium.

Sanman L.E., Chen I.W., Bieber J.M., Steri V., Trentesaux C., Hann B., Klein O.D., Wu L.F, & Altschuler S.J. (2021). Transit-amplifying cells coordinate changes in intestinal epithelial cell type composition. Developmental cell, 56(3), 356-365.e9.

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